The contamination of water and pastures can cause different diseases in the animals drinking the contaminated water and feeding on the pastures. The control of these possible sources of infection requires knowing if the pastures and water is contaminated in order to treat them accordingly, thus avoiding or reducing the risk of appearance of human or animal infections. The sources of such contamination include animal faeces, which may carry different organisms that could potentially contaminate the environment and/or pathogens affecting plants and animals, including humans.
Faecal microbiological contamination in water is usually analysed by determining the presence of organisms, not necessarily pathogenic, identified as “indicator or marker organisms”. In this respect, some faecal bacteria, such as Escherichia coli have been used as faecal contamination markers (EP 438115 A2).
Surprisingly, it has now been discovered that enteric viruses, which infect the gastro-intestinal tract and are excreted in the faeces, can be used as environmental contamination markers, in particular, to identify faecal contamination of animal origin affecting pastures and water.
Enteric viruses form a heterogeneous group including representatives of different virus families, of which the most important are possibly picornaviruses (basically, enteroviruses and teschoviruses), caliciviruses, astroviruses and reoviruses (rotaviruses) and hepatitis E virus. In spite of their heterogeneity, these viruses have a series of characteristics in common, since they are nearly all small RNA viruses without a membranous envelope and stable in an acid pH (the only DNA virus in the group is the adenovirus). They can therefore withstand their route through the gastrointestinal tract, in the epithelium of which they usually establish acute infections, causing gastro-enteritis. They are disseminated through the faeces and transmitted through the faecal-oral route. Because of their characteristic resistance to adverse environmental conditions, they remain in the environment for long periods, and water contaminated by faeces and the food in contact with such water, mainly molluscs, are their primary transmission carriers.
Of the enteric viruses, one of the largest and best known groups is that of the enteroviruses, some of them important pathogens for humans and animals. Enteroviruses (EV) belong to the Picornaviridae family and their genome consists of a simple chain RNA positive pole molecule (ssRNA+) with approximately 7,400 nucleotides (nt). These are the most common viruses and infect a large variety of mammals. Approximately 90 serotypes have been identified, 62 of which infect humans and 27 animals. Bovine enteroviruses (BEV) are broadly distributed all over the world and classified as enzootic in some countries. Two species have been described to date: BEV1 and BEV2 (Knowles, N. J., and Barnett, I. T. (1985), Arch Virol 83 (3–4), 141–55), although there are probably more that have not yet been identified. There are 3 species of porcine enteroviruses have been described. An ovine enterovirus (OEV-1) that is closely linked to the BEVs has been isolated. The existence of viruses has been described in ocean environments, although it is believes that most of the viruses found in natural water are cyanophages or other types capable of infecting microalgae. The existence of water contaminated by human enteric viruses has also been described, especially close to urban areas, related to sources of human infections [Chapron et al., 2000, Appl. Environ. Microbiol. 66:2520–2525; Abbaszadegan et al., 1993, Appl. Environ. Microbiol. 59:1318–1324; Abbaszadegan et al., 1999, Appl. Environ. Microbiol. 65:444–449; Bosch A., 1998, Int. Microbiol. 1:191–196; Pianetti et al., 2000, Epidemiol. Infect. 125:455–462; Schvoerer et al., Res. Microbiol. 152:179–186].
Another important group of porcine enteric viruses are Teschoviruses, related to enteroviruses (Zell, R., M. Dauber, A. Krumbholz, A. Henke, E. Birch-Hirschfeld, A. Stelzner, D. Prager, and R. Wurm. 2001. J Virol 75:1620–31) and including the agents causing both mild and severe forms of a neurological disease known as “Teschen-Talfan's disease”, in addition to reproductive failure, pneumonia, diarrhoea and skin lesions in pigs. It is assumed in general that each animal species is the natural host of a large number of enteroviruses, often enzootic, and many of them probably pending identification. Although it is not common, they do occasionally transfer from one species to another, and this often gives rise to new emerging viral diseases. This appears to have occurred in the case of swine vesicular disease, an important swine disease caused by a human enterovirus which adapted to swine some 50 years ago (Zhang, G., D. T. Haydon, N. J. Knowles, and J. W. McCauley. 1999. Journal of General Virology 800:639–51).
Other enteric viruses, which are often found contaminating surface water, are reoviruses, in particular rotaviruses. Like enteroviruses, this virus group is very large. There are six known rotavirus antigen groups (groups A-F), infecting a wide range of species. Each group has its own preferences. For instance, group A viruses have been isolated in most of the mammal and bird species studied, group B viruses infect humans, pigs, cows, sheep and rats, group C infects pigs and rarely humans, and groups D and F are typical of birds, whereas group E is usually found in swine. They are stable in water and highly resistant to changes in temperature, pH and salinity. This carrier that contaminated water and the molluscs living in such water are important transmission carriers of these pathogens, both by their consumption and from contact when bathing or during other recreational activities (Bos, P., M. Kirsten, R. E. Cronje, and A. D. Steele. 1995. S Afr Med J 85:887–91., Bosch, A. 1998. Int Microbiol 1:191–6). Rotaviruses are highly variable, often giving rise to new reassortant variants when two different variants infect the same host. These natural reassortants, which can arise from different animal species, could play a role in the appearance of emerging human and animal diseases (Vonsover, A., I. Shif, I. Silberstein, H. Rudich, Y. Aboudy, E. Mendelson, L. Shulman, T. Nakagomi, and 0. Nakagomi. 1993. J Clin Microbiol 31:1783–7). Recently, Gratacap-Cavallier et al have detected human and bovine rotaviruses in potable water in the south-east of France, during an epidemic outbreak of viral diarrhoea in children (Gratacap-Cavallier, B., O. Genoulaz, K. Brengel-Pesce, H. Soule, P. Innocenti-Francillard, M. Bost, L. Gofti, D. Zmirou, and J. M. Seigneurin. 2000. Appl Environ Microbiol 66:2690–2). The co-detection of pathogenic human and animal viruses in potable water suggests a possible zoonosis in this outbreak, with water acting as the source of propagation.
Astrovirus infections are considered to be emerging viral diseases. Like other enteric viruses, they are transmitted in faeces/orally and are very persistent in aquatic media. Outbreaks are associated to the consumption of molluscs contaminated with waste water, and the intake of water contaminated by faeces. Besides in humans, astrovirus strains have been isolated from different animal species, including cows, sheep, swine, cats, dogs, ducks and turkeys (Abad, F. X., R. M. Pinto, C. Villena, R. Gajardo, and A. Bosch. 1997. Appl Environ Microbiol 63:3119–22. Chapron, C. D., N. A. Ballester, and A. B. Margolin. 2000. J Appl Microbiol 89:11–5).
Caliciviruses are well-known viral diarrhoea agents in many species, including humans, cats, pigs, cows and minks, and also in a large number of marine mammals, which apparently act as natural reserve stores (Smith, A. W., D. E. Skilling, N. Cherry, J. H. Mead, and D. O. Matson. 1998. Emerg Infect Dis 4:13–20., Smith, A. W., D. E. Skilling, and S. Ridgway. 1983. J Am Vet Med Assoc 183:1223–5). They are divided into five strains, of which four (Sapporo, Norwalk, hepatitis E and marine caliciviruses) are well-known pathogenic agents in humans. Many of these diseases can be considered cases of zoonosis, and species transfers are not uncommon, as can be seen with the swine disease known as swine vesicular exanthema, caused by a calicivirus that can not be distinguished from the San Miguel Sea Lion virus (Smith, A. W., D. E. Skilling, and S. Ridgway. 1983. J Am Vet Med Assoc 183:1223–5). Outbreaks of gastro-enteritis caused by caliciviruses are associated to the consumption of water contaminated with faecal waste and bathing in contaminated water (Lodder, W. J., J. Vinje, R. van De Heide, A. M. de Roda Husman, E. J. Leenen, and M. P. Koopmans. 1999. Appl Environ Microbiol 65:5624–7). Furthermore, there is increasing evidence that hepatitis E, caused by a virus that is closely liked to caliciviruses, could be of a zoonotic nature, because this virus is also pathogenic in several animal species, both domestic and wild, and it is found serologically in a growing number of animals, including pigs (Balayan, M. S. 1997. J Viral Hepat 4:155–65).
Finally, the adenovirus group, also a virus of between 60 and 90 nm with no membranous envelope, which makes it stable in ambient conditions, has a double DNA chain genome. Although this virus is not characteristic of gastrointestinal infections, it can have similar symptoms, such as gastro-enteritis, and be excreted in the faeces of infected individuals. It is often found contaminating water and molluscs (Pina, S., M. Puig, F. Lucena, J. Jofre, and R. Girones. 1998. Appl Environ Microbiol 64:3376–82).
Breakthroughs in molecular biology techniques have led to highly sensitive and specific RT-PCR protocols (reverse transcription-polymerase chain reaction) with better performances than classical isolation methods for the detection of enteric viruses with an RNA genome such as enteroviruses. Besides being highly sensitive, RT-PCR reactions provides an ADN fragment that can be sequenced and used to typify the isolated virus. Detection generally uses specific RNA region indicators containing conserved motives shared by this group of viruses (Hyypia et al., 1989, J. Gen. Virol. 70:3261–3268; De León et al., 1990, Pro. 1990 Amer. Water Works Assoc. WQTC; Reynolds et al., 1996, Appl. Environ. Microbiol. 62:1424–1427; Puig et al., 1994, Appl. Environ. Microbiol. 60:2963–2970; Schwab et al., 1995, Appl. Environ. Microbiol. 61:531–537).